Background/Purpose:

Genetic, in vitro and in vivo evidence strongly implicate the activation of nucleic acid sensing toll like receptors (TLR) 7, 8, and 9 in the pathophysiology of systemic lupus erythematosus (SLE).  IRAK4 is a serine/threonine kinase activated by TLRs that utilizes the MyD88 adaptor protein for signaling.  The relative importance of IRAK4 scaffolding and kinase functions in signaling is not clear.  Indeed, at least one recent report indicates profound differences between species, stimuli, and cell types with regard to the requirement of IRAK4 kinase activity for cell activation[1]. Utilizing a potent, selective and cell-permeable small molecule inhibitor of IRAK4, we queried the importance of IRAK4 kinase activity in primary human cell based assays utilizing SLE disease relevant stimuli.

Methods:

Sera from SLE patients was screened for the ability to induce interferon-alpha (IFN-α) protein release from primary human plasmacytoid dendritic cells (pDC).  Immunoglobulin-G (IgG) was purified from SLE sera that could induce IFN from pDCs, and then combined with debris from apoptotic U937 cells as a source of TLR 7, 8 and 9 ligands, to form SLE immune complexes (SLE-IC). Human peripheral blood mononuclear cells (PBMC) were stimulated with SLE-IC, and type I interferon (i.e., IFN-α) release in cell culture supernatant was assayed by ELISA. Type I IFN exposure can be monitored in whole blood from SLE patients using an IFN-responsive gene signature, so the expression of IFN-induced genes was assayed in RNA from SLE-IC stimulated PBMC using quantitative real-time polymerase chain reaction (qRT-PCR). As auto-antibody production by B cells is also a key component of pathophysiology in SLE, B cell activation and differentiation induced by TLR ligands was measured by flow cytometry, and cytokine release by B cells was measured by ELISA.

Results:

The IRAK4 inhibitor potently blocked SLE-IC induced type I IFN release and IFN gene signature in PBMC. This compound also blocked B cell activation- B cell surface activation marker expression in response to R848 in whole blood, 7 day plasma cell differentiation in PBMC, and cytokine expression by purified B cells exposed to IFN-α and R848.

Conclusion:

Using a potent and selective inhibitor of IRAK4 kinase activity in primary human cells, we can demonstrate that IRAK4 kinase activity is essential to inflammatory cytokine release, type I IFN production, and B cell activation, all of which are components of SLE pathophysiology in vivo.

1.            Chiang, E.Y., X. Yu, and J.L. Grogan, Immune Complex-Mediated Cell Activation from Systemic Lupus Erythematosus and Rheumatoid Arthritis Patients Elaborate Different Requirements for IRAK1/4 Kinase Activity across Human Cell Types. The Journal of Immunology. 186(2): p. 1279-1288.

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ACR Meeting Abstracts - https://acrabstracts.org/abstract/toll-like-receptor-7-8-and-9-activation-of-primary-human-cells-by-lupus-immune-complexes-is-dependent-on-interleukin-1-receptor-associated-kinase-4-activity/